Vibrio cholera is the causative agent of cholera, a severed diarrheal illness that affects more than 7 million people worldwide each year. The pathogenesis of V. cholera infecting as been extensively studied, but important gaps in our understanding still exist. One of these gaps is the role of iron V. cholera pathogenesis. The long-range goals of this project are to characterize iron-regulated virulence genes in V. cholera and to use knowledge of these genes and their in vivo regulation to improve V. cholera vaccine development. The SPECIFIC AIMS designed to achieve these goals are: 1) further analysis of the role of IrgA in the pathogenesis of V. cholera infecting, including a role in iron uptake in vivo from lactoferrin, transferrin or ferrous iron, a role as an intestinal adhesion, or a role in resistance to bacterial killing in tensional secretions; 2) examination of the role of IrgB in the regulation of virulence genes, including the recently described heme uptake system of V cholera, using two-dimensional protein gel electrophoresis and analysis of TnphoA fusions; 3) identification and characterization of additional iron-regulated genes in V. cholera that relate to virulence in an animal model; 4) investigation of the overlap of the fur gene and iron in the regulation of genes involved in V. cholera pathogenesis, including determination of the virulence of a fur mutant and of mutants in genes negatively regulated by iron independently of Fur, positively regulated by iron, and negatively regulated by iron but only expressed in a fur mutant; 5) identification of iron-regulated proteins in V. cholera that are expressed in vivo and immunogenic, using immunoblotting with convalescent sera from patients with cholera, as well as sera and intestinal fluids from rabbits experimentally infected with V. cholera.